Self‐Splittable Transcytosis Nanoraspberry for NIR‐II Photo‐Immunometabolic Cancer Therapy in Deep Tumor Tissue

Abstract Cancer photo‐immunotherapy (CPIT) as an ideal strategy can rapidly release hostile signals by appropriate dosage of focal laser irradiation to unmask primary tumor immunogenicity and can activate adaptive immunity to control distant metastases. However, many factors, including disordered immunometabolism, poor penetration of photothermal agents and immuno‐regulators, inadequate laser penetration into the deep tumor region, restrict the therapeutic outcomes of CPIT. Here, a second near‐infrared window (NIR‐II) photo‐immunometabolic cancer therapy (PICT) by a programmed raspberry‐structured nanoadjuvant (PRNMT) is presented that can potentiates efficient immunogenic cell death (ICD) in deep tumor tissue and alleviates immunometabolic disorder. The PRNMT is architected through self‐assembly of indoleamine 2,3‐dioxygenase 1 (IDO‐1) inhibitor modified small‐sized CuS nanoparticles (CuS5) and tumor microenvironment (TME) responsive cationized polymeric matrix. The TME can trigger the splitting and surface cationization of PRNMT into small cationized CuS5 that feature high transcytosis potential and TME immunometabolic regulation. Upon NIR‐II irradiation, CuS5 induce homogeneous ICD and release immunometabolic regulator in deep tumor tissues, which ameliorates IDO‐1 mediated immunometabolic disorder and further suppresses regulatory T cells infiltration. PRNMT mediated PICT effectively delays the primary murine mammary carcinoma 4T1 tumor growth and inhibits the lethal pulmonary metastasis in combination with programmed cell death protein 1 (PD1) blockade.


Preparation of 5 nm CuS nanoparticles.
Small-sized CuS nanoparticles (5 nm) was prepared according to previous report. 2 After purifying by precipitating in acetone, the OAm modified CuS nanoparticles were stored in chloroform for next use. PAE-r-PEMAL/MT or PCM-r-PEMAL/MT polymers were further modified on the surface of CuS nanoparticles by ligand exchange procedure (denoted as CuS 5 ) at 1:1 mass ratio between CuS nanoparticles and relative polymer. The CuS 5 was dispersed in THF for further use.

Preparation of PRNs MT and NRNs MT .
Programmed raspberry-structured nanoadjuvant (defined as PRN MT ) containing CuS 5 and mPEG-b-PAE polymer were prepared. Typically, mass ratio of 3:1 between CuS 5 and mPEGb-PAE was resolved in 1 mL THF followed by addition of 5 mL Milli-Q water by syringe pump at a rate of 10 mL/h. The resulting PRN MT was dialyzed in water for removing THF.
Similar method was applied for NRN MT preparation. PRN and NRN were prepared by the CuS 5 and mPEG-b-PAE or mPEG-b-PCM with similar procedures.

Release Profile of 1-MT.
The release profile of 1-MT prodrug from the PRN MT were determined by dialysis method.
Briefly, PRN MT (1 mL) were transferred into a membrane tube, and 15 mL of phosphate buffer (pH 6.7) was used as the media. The samples were incubated at 37 ℃ after irradiation for 5 min or not and were incubated at 37 ℃. The media (1 mL) was moved out and the fresh media was added in the designed time intervals. Subsequently, the amount of 1-MT prodrug released from the PRN MT suspension was characterized by UV spectrophotometer (Abs at 280 nm).

Photothermal conversion of PRNs.
The photothermal conversion efficiency was calculated by the formula below. Detailed calculation was given as following: h is the heat transfer coefficient, s is the surface area of the container, and the value of hs is obtained from the Eq. 4 and Figure S8. In vitro cytotoxicity assay of PRNs evaluated by dead and live staining.
To evaluate the photothermal cytotoxicity of PRNs on 4T1 cells, we further visualized the cell condition after 1064 nm laser irradiation (1 W/cm 2 ). 4T1 cells were seeded on 24-well plates with a density of 5 × 10 4 cells per well. After incubating for 12 h, various formulations were added into 24-well plates for 4 h incubation. The cells were irradiated by NIR-II laser for 5 min or not and further be stained by Calcein-AM/PI after 4 h (the dilution ratio of Calcein-AM/PI is 1:500).

Colocalization of rab11 and PRNs.
4T1 cells were seeded on coverslips in a 24-well plate overnight (5 × 10 4 cells per well). Cells were then incubated with fresh medium containing PRNs at 37 °C. After incubation for 4 h, the cells were fixed with 4% formaldehyde at room temperature for 15 min and washed for 3 times. Cells were stained with rab11 rabbit monoclonal antibody (dilution 1:100) (Cell Signaling Technology, MA, USA) at 4 °C for 12 h and followed with goat anti-rabbit FITC conjugated IgG-HRP (Beyotime Biotechnology, Jiangsu, China) for 90 min incubation to visualize the rab11. Cell nuclei were stained with DAPI.

Transcytosis of PRNs from adhered cells to suspending cells.
4T1 cells (5 × 10 4 cells per well) were cultured in a confocal dish for 12 h. At pH 6.7, PRNs/Cy5 and NRNs/Cy5 were added and incubated for 4 h, following with Hoechest 33342 addition, respectively. The dishes were washed with PBS for three times, and then 1 mL of fresh culture medium containing fresh 2 × 10 4 suspended 4T1 cells pretreated with Hoechest 33342 was added. The cells were under real time observation using a laser confocal scanning microscope (CLSM, Nikon ECLIPSE Ti2, Tokyo, Japan) nd the time-lapsed images were recorded.

4T1 multicellular spheroids.
In detail, 5 mL 1.5% hot agarose solution was coated T25 flask and cooled to room temperature naturally. 4T1 cells (2 × 10 6 ) were added to the flask and incubated with 10 mL DMEM medium for 6 days to grow into spheroids. The spheroids were transferred to 6-well plates and incubated at pH 6.7 or pH 7.4 DMEM medium. Meanwhile, the spheroids were incubated with PRNs/Cy5 and NRNs/Cy5 (100 μg/mL, CuS concentration calculated by ICP-MS30020) for 4 h. After collecting and washing with PBS for 3 times. The spheroids were observed with CLSM Z stack method with 405 and 640 nm wavelength channels.
Tumor penetration of PRNs.

Flow cytometry assay of cell surface markers.
After 10 days from first tumor treatment, the mice were sacrificed and spleens were excised.
The spleen pieces obtained for single-cell analysis were gently meshed though nylon mesh.

Immunohistochemical and immunofluorescence analyses.
At the end of therapy, the tumor tissues excised from sacrificed mice were fixed in 4% formaldehyde for 24 h and embedded in paraffin. The tumor tissues slices were observed after H&E staining. For immunofluorescence staining, tumors were excised for frozen section at 10 th day post-irradiation treatment.